变异乙型肝炎表面抗原在毕赤酵母中的克隆表达及表面抗原诊断试剂的评价
发布时间：2018-07-21 12:48
【摘要】： 乙型肝炎是一种对公共健康危害严重的疾病。近年来随着乙型肝炎疫苗和抗病毒药物的广泛使用，乙型肝炎病毒的感染和传播得到了有效的防止。但是，乙型肝炎的防治是一项长期而艰巨的任务，随着对乙肝病毒研究的深入，一些新的问题不断被发现，，如免疫后人群，抗病毒药物治疗的患者中以及慢性HBV感染的人群中都出现了HBV表面抗原的变异。这些变异对免疫诊断试剂提出了新的挑战。大量文献报道了诊断试剂对变异表面抗原的漏检。但是国内对HBV变异的流行病学及变异对国产诊断试剂影响的研究还较少。 本研究第一部分利用已构建好的野生型及3个HBV S区变异的质粒(T126N，D144A，G145R)作为模板，设计含有特定酶切位点的引物，进行PCR后用EcoRⅠ和NotⅠ双酶切回收的PCR产物，连接入酵母表达质粒pPICZA和pPICZα，构建出HBV S区野生及变异酵母表达质粒。测序结果显示，读码框架及核苷酸序列均正确。 利用SalⅠ酶切构建好的6种表达质粒(胞内表达4种，分泌表达2种)使之成为线性片断。电穿孔转化P．pastroris酵母细胞GS115，用高浓度抗生素Zeocine(2000ng／ml)加压筛选出高效表达的含有多拷贝整合的菌株。 对于筛选出的高抗性的菌株，经甲醇诱导表达后，用玻璃珠破碎法裂解酵母细胞，释放菌体内的表达产物。各菌株的裂解液稀释相同的倍数，用雅培公司的HBsAg试剂ARCHETECT对表达的HBsAg定量，并利用确证试剂进行确证，结果均为阳性。选择表达量高的菌株进行大量表达。表达产物以多克隆抗体作为一抗，使用免疫印迹法(Western blot，WB)检测4种表达产物，结果证明表达产物为HBsAg。 本研究的第二部分是利用各种HBsAg对国内外诊断试剂进行评价。为了确保实验结果的可靠性，排除由于实验操作问题对结果的影响，我们对ELISA实验操作进行了规范，选择了一份HBV表面抗原强阳性血清倍比稀释后，作为检测用血样。利用各家诊断试剂对血样于不同时间进行了10次检测，最后结果统计显示试验的重复性及精确性都己达到要求。并且，在以后利用变异抗原对诊断试剂进行评价时，仍然将此血清作为内标以控制试验操作的一致性。 将酵母表达的抗原和我们收集到的CHO细胞和小鼠L细胞表达的抗原稀释到一定的浓度，用各种HBsAg诊断试剂进行检测，对国内外诊断试剂进行评价。结果发现国产诊断试剂对HBV野生型不同亚型的检测灵敏度与进口试剂相差了约4-32倍。而对多种不同变异抗原的检测，4家国产试剂均存在一定的漏检情况。并且，利用不同表达系统表达的变异抗原对诊断试剂的评价结果也是基本相同的。国产试剂对G145R变异及其相关的多点变异的检测能力弱；对T118K／P120Q，Q129R／M133T等联合变异的检测能力亦有待于提高；而对于T126N，D144A等单点变异国内外试剂的检测能力基本一致。同时，用含有1／4 G145R变异的血样对诊断试剂进行评价，发现与以上相同的结论，国产试剂的检测灵敏度下降。
[Abstract]:Hepatitis B is a serious disease to public health. With the widespread use of hepatitis B vaccine and antiviral drugs in recent years, the infection and transmission of hepatitis B virus have been effectively prevented. However, the prevention and control of hepatitis B is a long-term and arduous task. With the in-depth study of HBV, some new types of hepatitis B virus have been studied. Problems have been discovered, such as the immunization of the population, in the patients with antiviral drugs and of the HBV surface antigen variation in the people with chronic HBV infection. These variations pose a new challenge to the immunodiagnostic reagents. A large number of documents have reported the leakage of the diagnostic reagents to the variant surface antigenic agents. However, the epidemic of HBV variation in China has been reported. There are few studies on the effects of learning and mutation on domestic diagnostic reagents.
In the first part of this study, using the constructed wild type and 3 HBV S region variant plasmids (T126N, D144A, G145R) as a template, the primers containing specific enzyme cut sites were designed and the PCR products recovered by EcoR I and Not I were reclaimed after PCR. The yeast expressed plasmid pPICZA and pPICZ alpha were connected to the yeast expression plasmid pPICZA and pPICZ alpha, and a wild and variant yeast table was constructed. Plasmid sequencing and sequencing showed that the coding frame and nucleotide sequence were correct.
6 kinds of expression plasmids (4 kinds of intracellular expression and 2 kinds of secretory expression) formed by Sal I enzyme were used as linear fragments. Electroporation was used to convert P.pastroris yeast cell GS115, and high concentration antibiotic Zeocine (2000ng / ml) was used to screen the highly expressed strains with multi copy integration.
After the screening of highly resistant strains, the yeast cells were cleaved by glass bead breakup and the expression products were released by glass bead breakage. The lysate of each strain was diluted the same multiplier. The expression of HBsAg was quantified by the HBsAg reagent ARCHETECT of the Abbott Company and confirmed by the reagents. The results were all positive. Western blot (WB) was used to detect 4 kinds of expression products, and the result showed that the expression product was HBsAg..
The second part of this study is to evaluate the domestic and foreign diagnostic reagents using various HBsAg. In order to ensure the reliability of the experimental results and eliminate the effect of the experimental operation on the results, we have standardized the experimental operation of ELISA, and selected a HBV surface antigen strong positive serum double dilution, as a detection blood sample. 10 tests were carried out on the blood samples at different times. The results of the results showed that the repeatability and accuracy of the test had reached the requirement. And, when the diagnostic reagent was evaluated by using the variant antigen, the serum was still used as the internal standard to control the consistency of the experiment.
The antigen expressed by yeast and the antigen expressed by the CHO and L cells of the mice were diluted to a certain concentration, and the diagnostic reagents were evaluated by various HBsAg diagnostic reagents. The results showed that the detection sensitivity of the domestic diagnostic reagents to the different subtypes of HBV wild type was about 4-32 times that of the imported reagents. The 4 homemade reagents were detected by 4 homemade reagents, and the results of the diagnostic reagents expressed by different expression systems were basically the same. The ability of the domestic reagents to detect the variation of G145R and its related multipoint mutations was weak; T118K / P120Q, Q129R / M133T, etc. The detection ability of the mutation is still to be improved, and the detection ability of the domestic and foreign reagents of T126N, D144A and other single variation is basically the same. At the same time, the diagnostic reagents are evaluated with the blood samples containing 1 / 4 G145R variation, and the same conclusion is found.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
[Abstract]:Hepatitis B is a serious disease to public health. With the widespread use of hepatitis B vaccine and antiviral drugs in recent years, the infection and transmission of hepatitis B virus have been effectively prevented. However, the prevention and control of hepatitis B is a long-term and arduous task. With the in-depth study of HBV, some new types of hepatitis B virus have been studied. Problems have been discovered, such as the immunization of the population, in the patients with antiviral drugs and of the HBV surface antigen variation in the people with chronic HBV infection. These variations pose a new challenge to the immunodiagnostic reagents. A large number of documents have reported the leakage of the diagnostic reagents to the variant surface antigenic agents. However, the epidemic of HBV variation in China has been reported. There are few studies on the effects of learning and mutation on domestic diagnostic reagents.
In the first part of this study, using the constructed wild type and 3 HBV S region variant plasmids (T126N, D144A, G145R) as a template, the primers containing specific enzyme cut sites were designed and the PCR products recovered by EcoR I and Not I were reclaimed after PCR. The yeast expressed plasmid pPICZA and pPICZ alpha were connected to the yeast expression plasmid pPICZA and pPICZ alpha, and a wild and variant yeast table was constructed. Plasmid sequencing and sequencing showed that the coding frame and nucleotide sequence were correct.
6 kinds of expression plasmids (4 kinds of intracellular expression and 2 kinds of secretory expression) formed by Sal I enzyme were used as linear fragments. Electroporation was used to convert P.pastroris yeast cell GS115, and high concentration antibiotic Zeocine (2000ng / ml) was used to screen the highly expressed strains with multi copy integration.
After the screening of highly resistant strains, the yeast cells were cleaved by glass bead breakup and the expression products were released by glass bead breakage. The lysate of each strain was diluted the same multiplier. The expression of HBsAg was quantified by the HBsAg reagent ARCHETECT of the Abbott Company and confirmed by the reagents. The results were all positive. Western blot (WB) was used to detect 4 kinds of expression products, and the result showed that the expression product was HBsAg..
The second part of this study is to evaluate the domestic and foreign diagnostic reagents using various HBsAg. In order to ensure the reliability of the experimental results and eliminate the effect of the experimental operation on the results, we have standardized the experimental operation of ELISA, and selected a HBV surface antigen strong positive serum double dilution, as a detection blood sample. 10 tests were carried out on the blood samples at different times. The results of the results showed that the repeatability and accuracy of the test had reached the requirement. And, when the diagnostic reagent was evaluated by using the variant antigen, the serum was still used as the internal standard to control the consistency of the experiment.
The antigen expressed by yeast and the antigen expressed by the CHO and L cells of the mice were diluted to a certain concentration, and the diagnostic reagents were evaluated by various HBsAg diagnostic reagents. The results showed that the detection sensitivity of the domestic diagnostic reagents to the different subtypes of HBV wild type was about 4-32 times that of the imported reagents. The 4 homemade reagents were detected by 4 homemade reagents, and the results of the diagnostic reagents expressed by different expression systems were basically the same. The ability of the domestic reagents to detect the variation of G145R and its related multipoint mutations was weak; T118K / P120Q, Q129R / M133T, etc. The detection ability of the mutation is still to be improved, and the detection ability of the domestic and foreign reagents of T126N, D144A and other single variation is basically the same. At the same time, the diagnostic reagents are evaluated with the blood samples containing 1 / 4 G145R variation, and the same conclusion is found.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
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相关期刊论文 前9条
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